Lecture 6

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25 Mar 2012
Lecture 6: DNA Replication (Part 2)
1. Issues in replication
2. DNA repair
A quick key word review
oLeading strand is synthesized continuously from single RNA primer
oLagging strand is synthesized discontinuously from multiple primers
oOkazaki fragments consist of the RNA primer and the DNA strand
oDNA synthesis proceeds in 5’ to 3’ direction
oPrimosome consists of primase and helicase
oThe predominant helicase is on lagging strand
Issues in DNA replication
oWhat happens at the ends of eukaryotic linear chromosomes during replication
oHow is DNA unwound?
oHow are mistakes found and corrected?
What happens at the ends of chromosomes?
oAs you migrate through the double stranded DNA, particularly in lagging
strand which is initiated by primers, the primers run out of template
oThe leading strand rolls along happily
The problem at ends of chromosomes
oThe primer space is programmed by primosome to space them in specific
oOver successive rounds of replication, a gradual shortening of 5’ ends of the
newly synthesized strands, particularly in lagging strands – you lose sequence
What happens at the ends?
oAdds repeated sequences in the 3’ ends of strands which allows the replication
machinery to faithfully reproduce
oThe ends are called telomeres and they provide access points for primers to be
oThe t-loops are usually tucked back into the chromosome. The reason for loop-
like structure is that cells don’t like to see chromosomes with single stranded
DNA strung out at the end, because they’ll think it’s a broken chromosome and
they’ll do their best to eliminate them
telomerase to the rescue
oThe enzymes which adds these 3’ extensions is called telomerase and it’s able
to add nucleotides to the 3’ ends of the DNA in the absence of a primer,
because it has its own built-in primer and it uses the RNA molecule template
for the addition of nucleotides to the 3’ end
oBecause it’s not a long stretch of RNA, you get a small stretch of RNA as a
template that is repeated a lot of times. So you get a G-rich strings being
added to the 3’ end which is using multiple copies using this template
Telomere replication
oWith RNA template you end up with complimentary copies added to the
oTelomerase binds and recognized the 3’ end with complimentary base pairing.
Now it starts to add the replicates of this particular complimentary sequence to
the RNA template
oDepending on the cell type, a small rounds or very many rounds of additions
oThe typical DNA polymerase can then establish a primer in a particular location
and continue using DNA polymerase and complete the sequence
oThese type of enzymes essentially resemble a reverse transcriptase (a type of
polymerase which is able to add nucleotides)
oThis telomerase generates G-rich ends and adds nucleotides to 3’ ends to
parental strands template
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oThis means that 3’ ends are all significantly longer than the 5’ ends. Even
though the 3’ ends are a bit folded back, there are some unbalance between
the 5’ and 3’ ends
Homeostatic control of telomere length
oThe cell is using telemetric addition to maintain replication fidelity for entire
length of the chromosome, so if telomeric sequence isn’t added back on again
after each rounds of replication, the chromosome will shorten
oOn the other hand, if the cell adds a certain number of them back on after
each round of replication, it provides a clock mechanism for the cell
As I get older, my telomerase activity’s now going to diminish and now I’m
going to add fewer and fewer of these telomeric sequences after each
round of replication. And ultimately, my telomerase activity will become so
incompensatated that I will start to lose important sequence information
oCells have a mechanism where it can modulate the telomere length and it’s an
on-board replication to stop extensive/continuous replication that might go out
of control (sort of what we define cancer as)
Telomeres and cancer
oIf you look at embryonic or stem cells, you see extensive telomeric lengths
indicating these cell lines are the ones that has to be replicated over an
extended period of time and they want to maintain their telomeric sequences
as long as possible
oSomatic cells only need to be around for a defined period of time, and then
they go into replicative cell synasis
After defined period of replications of cell divisions, the cells lose capacity
to continue to divide
We have limited replications for somatic cells
When they get to the point where they really can’t do anything more, they
look at their telomeric lengths and shrink
Apoptosis: cell suicide the cell sees the shortening of the telomeres
to the unacceptable length, and that initiates the suicide (chromosome
breakage, destruction of the anterior of the cell
oExceptionally long telomeres: persistent growth, no programmed cell death,
cancer cells
oMost cancer cells produce high levels of telomerase
oModification of the telomerase RNA template interferes with cancer cell growth
oPrognoses of some cancers can be ascertained by telomerase levels
oCell-targeted inhibitors of telomerase activity have been suggested as
therapeutic agents
The winding problem
oHelicase is capable of separating strands but ultimately all that coiling begins
to build up within the helix itself and start to form knots and super coils where
the DNA becomes twisted to itself simply because the tortional stress imparted
by pulling the strands apart
oSo eventually the helicase cannot go any further because of the pressure,
stress imparted by the super coiled double helix it’s bumping into
Stress released by topoisomerase type 1
oThe enzyme involved in this is called topoisomerase and it’s able to initiate a
nick in 1 strand of the DNA and that allows for free rotation of DNA around that
single access that remains and this eliminates the torsional stress on DNA
oThe type of break involved is single stranded break cuts the phosphodiester
linkage in one side
oThis allows DNA to freely rotate around the sugar phosphate backbone of the
remaining strand
oThe free hydroxyl that is attached to tyrosine, which is the R group of
topoisomerase type 1, is able to initiate a tack on the phosphodiester linkage
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